U.S. patent application number 11/409927 was filed with the patent office on 2007-01-04 for methods for the treatment of multiple myeloma.
Invention is credited to Jean-Marc Gauguet, Irina Mazo, Ulrich H. von Andrian.
Application Number | 20070003558 11/409927 |
Document ID | / |
Family ID | 37215330 |
Filed Date | 2007-01-04 |
United States Patent
Application |
20070003558 |
Kind Code |
A1 |
von Andrian; Ulrich H. ; et
al. |
January 4, 2007 |
Methods for the treatment of multiple myeloma
Abstract
Methods for treating multiple myeloma with inhibitors of CXCR4
are described. The decreased expression of CXCR4 on multiple
myeloma cells according to the invention results in decreased
homing of the cells to the bone marrow and a reduction in the
development of the disease. Also disclosed are pharmaceutical
compositions incorporating such inhibitors for use in the
therapeutic treatment of multiple myeloma. The treatment methods
described herein can be used independently, or in conjunction with,
other therapies for the treatment of multiple myeloma.
Inventors: |
von Andrian; Ulrich H.;
(Brookline, MA) ; Mazo; Irina; (Chestnut Hill,
MA) ; Gauguet; Jean-Marc; (Boston, MA) |
Correspondence
Address: |
GOSZ AND PARTNERS, LLP
450 BEDFORD STREET
LEXINGTON
MA
02420
US
|
Family ID: |
37215330 |
Appl. No.: |
11/409927 |
Filed: |
April 24, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60674463 |
Apr 25, 2005 |
|
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Current U.S.
Class: |
424/155.1 ;
514/44R |
Current CPC
Class: |
C12N 2799/027 20130101;
A61K 31/00 20130101; A61K 39/395 20130101; A61K 45/06 20130101 |
Class at
Publication: |
424/155.1 ;
514/044 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 39/395 20060101 A61K039/395 |
Goverment Interests
GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT
[0002] This work was funded in whole or in part by a grant from the
National Institutes of Health pursuant to Grant No. HL56949. The
federal government may have certain rights in the invention.
Claims
1. A method for treating a subject having, or at risk of
contracting, a hematological malignancy comprising administering to
the subject an effective amount of a pharmaceutical composition
comprising an inhibitor for the CXCR4 receptor.
2. The method of claim 1 wherein the hematological malignancy is a
malignancy of the bone marrow.
3. The method of claim 2 wherein the malignancy involves malignant
cells expressing the CXCR4 receptor.
4. The method of claim 1 wherein the hematological malignancy is
multiple myeloma.
5. The method of claim 1 wherein the inhibitor interferes with or
blocks an interaction between the CXCR4 receptor and the chemokine
SDF-1.alpha./CXCL12.
6. The method of claim 1 wherein the inhibitor is a small
molecule.
7. The method of claim 1 wherein the inhibitor is an antibody.
8. The method of claim 7 wherein the antibody is a monoclonal
antibody.
9. The method of claim 1 wherein the inhibitor is an siRNA moleucle
which inhibits the synthesis, post-translatioinal modification or
functioning of the CXCR4 receptor
10. The method of claim 1 wherein the subject is a human.
11. The method of claim 1 wherein the pharmaceutical compostion
includes a pharmaceutically acceptable carrier and an adjuvant.
12. The method of claim 1 which is used as an adjunct to another
therapuetic treatment.
13. The method of claim 12 wherein the other therapy is also used
for treating multiple myeloma.
14. A pharmaceutical composition for the treatment of a
hematological malignancy comprising an inhibitor for the CXCR4
receptor on a cell implicated in the malignancy, a
pharmacueticallyk acceptable carrier, and an adjuvant.
15. The pharmaceutical composition of claim 14 wherein the
hematological malignancy is multiple myeloma.
16. The pharmaceutical composition of claim 14 which is an oral
formulation.
17. The pharmaceutical composition of claim 14 which is targeted to
the bone marrow of a patient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority from U.S.
Provisional Application No. 60/674,463, filed on Apr. 25, 2005, the
specification of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] This invention relates to methods for the treatment of
hematological malignancies that may reside in the bone marrow,
particularly malignancies implicated by the expression of the CXCR4
receptor, such as multiple myeloma, using inhibitors of CXCR4.
CXCR4 is known to be the receptor for the chemokine
SDF-1.alpha./CXCL 12. The preferred inhibitors of this invention
are those which interfere with or block the CXCR4/SDF signaling
pathway in humans.
[0004] The CXCR4 receptor is typically found on hematopoietic cells
that reside in the bone marrow, such as white blood cells. The
CXCR4 receptor is also expressed on human multiple myeloma cells
and mouse multiple myeloma cells, i.e. cells isolated from mice
that spontaneously develop multiple myeloma, as well as a variety
of other cell types, such as stem cells. The expression of the
CXCR4 receptor on these cells is believed to be a factor in their
migration to the bone marrow, and for their retention in the bone
marrow, and for regulating the trafficking and homing of cells
involved in the immune system.
[0005] In humans and mice, multiple myeloma cells grow
preferentialy in the bone marrow. These cells enter the bloodstream
from their point of origin (typically a lymphoid tissue or the bone
marrow itself). The cells inflitrate the bone marrow in previously
unaffected bones until the bone marrow in all bones of the body
become involved. The specific mechanism that allows these multiple
myeloma cells to traffic from the blood to the bone marrow has not
been elucidated as yet. See C. Moller et al., Leukemia, 17, pages
203-210 (2003).
[0006] Multiple myeloma is a form of cancer known to affect
approximately 50,000 patients in the United States, with
approximately 15,000 new patients diagnosed annually. Multiple
myeloma is a hematological malignancy characterized by the clonal
proliferation and accumulation of immunoglobulin-producing plasma B
cells in bone marrow, causing the progressive destruction of bone
tissue and bone marrow. If left untreated, the condition ultimately
leads to the death of the patient.
[0007] Current treatments for mutiple myeloma center on the
therapeutic use of various alkylating agents, anthracyclines and
corticosteroids. These treatments typically do not cure the
disease, but can effectively extend the life of the pateint for 3-4
years on average. Novel therpaies have also been proposed to target
not only multiple myeloma clls, but also multiple myeloma/host cell
interactions, as well as the bone marrow itself. These newer
therapies target mechanisms whereby multiple myeloma cells grow and
survive in the bone marrow, and utilize drugs such as thalidomide,
thalidomide derivatives, and proteasome inhibitors. See T.
Hideshima et al., Immunological Reviews, 194, pages 164-176
(2003).
[0008] A drug developed by AnorMed, Inc, designated as AMD3100,
originally developed as an HIV therapeutic, has been evaluated in
clinical trials for mobilizing stem cells that can be ultimately
used in indications such as multiple myeloma. AMD3100 is described
as a stem cell mobilizer that triggers the rapid movement of stem
cells out of the bone marrow and into circulating blood by blocking
the cellular receptor CXCR4. Once in the criculating blood of a
subject, the stem cells can be collected for use in stem cell
transplants in an attempt to restore the immune systenm of cancer
patients who have had other treatments that previously destroyed
immune cells. Typical drugs of this general description are
disclosed in U.S. Pat. No. 6,825,351, issued Nov. 30, 2004, and
U.S. Pat. No. 6,835,731, issued Dec. 28, 2004, the respective
disclosures of which are incorporated herein in their
entireties.
[0009] In spite of all the recent advances made in multiple myeloma
treament, the disease remains largely incurable due, in part, to
the development of tumor cell resistance to conventional therapies.
Accordingly, there is currently no known cure for the disease.
[0010] It is therefore an objective of this invention to provide
methods for the treatment of multiple myeloma, and other
hematological malignancies that are associated with the bone
marrow, using therapeutic treatments which are effective and are
not impeded by drug resistance. It is a further objective of this
invention to provide methods for the treatment of multiple myeloma
utilizing therapies directed to interrupting the CXCR4/SDF
signaling pathway of multiple myeloma cells in patients.
SUMMARY OF THE INVENTION
[0011] According to the present invention, a method for treating a
hemtological malignancy in a mammal is provided comprising
administering to the mammal an effective amount of a CXCR4
inhibitor. The CXCR4 inhibitor disrupts or impedes the CXCR4
binding to the chemokine SDF-1.alpha./CXCL12. The inhibitor is
administered to the mammal in need of such treatment as a
pharmaceutical composition in amounts effective to interrupt the
CXCR4-SDF pathway and to treat this disease. SDF-1.alpha./CXCR4
signaling is active in the majority of cancer cells, including
chronic lymphoid leukemia (CLL), chronic myeloid leukemia (CML),
acute myelogenous leukemia (AML), acute lymphoblastic leukemia
(ALL), and non-Hodgkin's lymphoma (NHL). In addition, this
signaling pathway is also active in solid tumors, such as
rhabdomyosarcoma, prostate cancer and melanoma. Breast and lung
cancers are known to metastasize to the bone marrow, using the
SDF-1.alpha./CXCR4 pathway in the process.
[0012] In one embodiment, the CXCR4 chemokine receptor is on a
multiple myeloma cell located in the bone marrow of a patient. The
inhibitor is part of a pharmaceutical composition, along with other
additives, and the composition is targeted to the bone marrow to
impede or prevent the spread of the disease. Suitable inhibitors
include antibodies to CXCR4, preferrably monoclonal antibodies,
small molecules, siRNA molecules, and dominant negative
inhibitors.
[0013] In another embodiment, the invention is a pharmaceutical
composition targeted to the bone marrow. The pharmaceutical
composition includes a phsyiologically acceptable carrier and an
adjuvant. The pharmaceutical compositon can be used indepentantly
of other therapeutic compositions, or in combination with such
other therapeutics.
[0014] The various features and advantages of the present invention
will be better understood from the following specification when
read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A-1B are graphs illustrating the expression of CXCR4
on (FIG. 1A) vector 5T33MM mouse cells and (FIG. 1B)
SDF-1.alpha.degrakine (SDF-DK) 5T33MM cells. In the Figures, the
red lines indicate the staining levels with control antibodies, and
the blue lines indicate the presence of staining with a CXCR4
antibody.
[0016] FIGS. 2A-2E are graphs illustrating the
homing/retention/proliferation/survival of control 5T33MM cells and
cells which are genetically modified to prevent surface CXCR4
expression in bone marrow.
[0017] FIG. 3 is a graph demonstrating the role of CXCR4 in the
homing/retention/proliferation/surviuval of 5T33MM cells when
CXCR4-CXCL12 interaction is inhibited pharmacologically.
[0018] FIG. 4 is a graph illustrating the survival of mice injected
with 5T33MM transduced with (a) a vector, or (b)
SDF-1.alpha.degrakine (SDF-DK) cells.
[0019] FIG. 5 is a graph illustrating the effects of AMD3100 and
PBS administered to mice injected with 10.times.10.sup.6
5T33.sup.eGFP cells.
DETAILED DESCRIPTION OF THE INVENTION
[0020] This invention provides a method for treating multiple
myeloma and other hematological malignancies of the bone marrow
which involve the expression of the CXCR4 receptor by affected
cells. The invention also provides a method for treating medical
disorders by interrupting the CXCR4/SDF signaling pathway in the
bone marrow cells of a mammal.
[0021] As used herein, the following terms and phrases shall have
the following meanings unless indicated otherwise.
[0022] A "subject", as used herein, includes mammals such as human
and non-human mammals. Preferred non-human mammals include
primates, pigs, rodents, rabbits, canines, felines, sheep, horses,
and goats. Veterinary applications are deemed within the scope of
the present invention.
[0023] The abbreviations "MM" and "BM", as occasionally used
herein, designate multiple myeloma and bone marrow,
respectively.
[0024] The terms "treatment" or "treating" a medical condition,
such as multiple myeloma or other hematological malignancies of the
bone marrow, are intended to include both prophylactic and
therapeutic methods of treating a subject. "Treatment" generally
denotes the administration of a therapeutic agent to a subject
having a disease or disorder, a symptom of a disease or disorder,
or a predisposition toward a disease or disorder, for the purpose
of preventing, alleviating, relieving, reducing the symptoms of,
altering, or improving the medical condition or disorder. The
methods of treatment described herein may be specifically modified
or tailored based on a specific knowledge of the subject obtained
by pharmacogenomics, and other methods for analyzing individual
drug responses to therapies.
[0025] By "vaccine" is meant a prophylactic compostion intended to
be used for the suppression, treatment or prevention of a disease.
Vaccines can be administered orally, intravenously, nasally or
subcutaneously.
[0026] An "inhibitor" in the context of the invention generally
denotes an agent that can inhibit the interaction between the CXCR4
chemokine receptor and its ligand, and particularly the chemokine
SDF-1.alpha./CXCL12 ("SDF"). By "inhibiting" an interaction is
generally meant, in the context of this invention, that the CXCR4
receptor and SDF chemokine are unable to properly bind to each such
that the signaling pathway is interrupted. Such inhibition can
result from a variety of events, such as by interrupting,
preventing or reducing the binding of the chemokine to the
receptor, inactivating the receptor or the chemokine, such as by
cleavage or other modification, altering the affinity of the
receptor and chemokine for each other, preventing or reducing the
expression of the CXCR4 receptor on a cell, expressing an abnormal
or inactive CXCR4 receptor, deactivating the CXCR4 receptor,
preventing or reducing the proper conformational folding of the
CXCR4 receptor, interfering with signals that are required to
activate or deactivate the CXCR4 receptor or the SDF chemokine, or
interfering with other molecules required for the normal synthesis
or functioning of the receptor or chemokine.
[0027] Examples of types of inhibitors useful in the present
invention are soluble forms of CXCR4 or SDF, inhibitory proteins,
such as antibodies, inhibitory peptides and proteins, inhibitory
carbohydrates, inhibitory glycoproteins, chemical entities, and
small molecules.
[0028] A purified form of either CXCR4 or SDF, or a portion or
fragment thereof, can compete with its cognate molecule for the
binding site on the complementary molecule, and thereby reduce or
eliminate binding between the CXCR4 receptor and the SDF chemokine.
The soluble form can be obtained by natural or recombinant means,
and is intended to include truncated forms and fragments.
[0029] Inhibitory proteins include anti-CXCR4 antibodies and
anti-SDF antibodies, including humanized antibodies, chimeric
antibodies, Fab.sub.2 antibody fragments, polyclonal antibodies,
and monoclonal antibodies. Monoclonal antibodies are preferred.
[0030] Inhibitory peptides include peptides or fragments of CXCR4
or SDF, which normally bind to CXCR4 so that the interaction or
binding between CXCR4 and SDF is reduced or eliminated. The
inhibitory peptides can recognize the binding site, or a portion of
the binding site, of CXCR4.
[0031] Chemical entities and small molecules which are designed to
interrupt the CXCR4/SDF pathway are also within the scope of the
invention.
[0032] A "therapeutically effective amount" of a pharmaceutical
composition means that amount which is capable of treating, or at
least partially preventing or reversing the symptoms of, the
medical condition or disease state. A therapeutically effective
amount can be determined on an individual basis and is based, at
least in part, on a consideration of the particular species of
mammal, for example, the mammal's size, the particular inhibitor
used, the type of delivery system used, and the time of
administration relative to the progression of the disease. A
therapeutically effective amount can be determined by one of
ordinary skill in the art by employing such factors and using no
more than routine experimentation.
[0033] Multiple myeloma can be characterized by a clonal
proliferation and accumulation of immunogolbulin-producing plasma B
cells in the bone marrow. Without intending to be bound by any
particualar theory or mechanism of action, it is believed that
malignant multiple myeloma cells may derive from B cells that have
undergone antigen selection in the germinal center of secondary
lymphoid organs. Such organs include the spleen and peripheral
lymph nodes. To reach the bone marrow, multiple myeloma cells are
also believed to home to the bone marrow using a multi-step
molecular pathway, in a manner analogous to that of other leukocyte
subtypes.
[0034] Blood-borne cells engage several sequential adhesion
pathways to leave the circulatory system and lodge in a target
organ. The initial steps of tethering and rolling are mediated by
primary adhesion molecules, such as selectins or .alpha.4
integrins. This initial interaction does not require specific
activation signals. To stop rolling and arrest, cells must engage
secondary receptors, represented by the integrin family (.beta.2 or
.alpha.4), which must be activated to mediate firm adhesion.
Integrin activation signals are frequently (but not always)
provided by chemokines that are present on the endothelial cell
surface. Most chemoattractant signals depend on G.alpha.i-coupled
(i.e. pertussis toxin (PTX)-sensitive) receptors. Activated
integrins interact with endothelial immunoglobulin superfamily
ligands. While some chemokines trigger intravascular adhesions,
others direct migration of the family adherent leukocytes into and
within the extravascular space. How adherent leukocytes "sense"
chemokine gradients within a tissue and follow them to reach their
end target is largely unknown.
[0035] Turning now to the Figures, FIGS. 1A-1B are a series of
graphs showing the results achieved when 5T33MM cells are
transduced with a control vector (FIG. 1A) or with an SDF-1.alpha.
degrakine (FIG. 1B). In the Figures, a red line indicates the
control antibody staining, and the blue line indicates the CXCR4
antibody staining.
[0036] FIGS. 2A-2E are a series of graphs illustrating the homing
of 5T33MM cells to the bone marrow, and the
survival/retention/proliferation of the 5T33MM cells at time
intervals of 1 hour, 24 hours, 1 week, 2 weeks and 3 weeks after
injection.
[0037] FIG. 3 is a graph demonstrating the
homing/retention/proliferation/survival of 5T33MM cells when
CXCR4-CXCL12 interaction is inhibited pharmacologically. AMD3100
and PBS is implanted, and the survival rate after 2 hours, 24 hours
and 6 days is measured.
[0038] FIG. 4 is a graph illustrating the results on mice injected
with 5T33MM cells transduced with a vector, and 5T33MM cells
transduced with SDF-1.alpha. (SDF-DK) cells. The survival of the
mice is measured using the Kaplan-Meier survival curve, P<0.0001
between the vector and SDF-DK by the log-rank test, wherein n=9 for
the vector, and n=11 for SDF-DK.
[0039] FIG. 5 is a graph showing the survival of C57BL/6 mice after
injection with 10.times.10.sup.6 5T33.sup.eGFP cells. Osmotic pumps
are installed at the time of injection, and either AMD3100 or PBS
is implanted. The osmotic pumps are changed every 7 days, and the
pump administration is discontinued after 21 days. The survival
rate, P<0.05, is measured between PBS and AMD3100 by the
log-rank test, n=3.
[0040] The CXCR4 inhibitors of this invention can be incorporated
into pharmaceutical compositions suitable for administration to a
subject. Such compositions typically comprise the inhibitor and a
pharmaceutically acceptable carrier. As used herein, the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifingal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media, and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, the use thereof in
the pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
present compositions.
[0041] The administration of the active compounds (CXCR4
inhibitors) of the invention may be for either a prophylactic or
therapeutic purpose. Accordingly, in one embodiment, a
"therapeutically effective dose" refers to that amount of inhibitor
sufficient to result in a detectable change in the physiology of a
recipient patient. In another embodiment, a "therapeutically
effective dose" refers to an amount of inhibitor sufficient to
result in modulation of the CXCR4/SDF pathway. In yet another
embodiment, a "therapeutically effective dose" refers to an amount
of inhibitor sufficient to result in the amelioration of symptoms
of multiple myeloma. In still another embodiment, a
"therapeutically effective dose" refers to an amount of inhibitor
sufficient to prevent the occurrence of multiple myeloma in a
patient.
[0042] Toxicity and therapeutic efficacy of the inhibitory
compounds of the invention can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index, and it can be
expressed as the ratio LD.sub.50/ED.sub.50. Compounds which exhibit
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
the affected tissue, i.e. the bone marrow in most instances, in
order to minimize potential damage to uninfected cells, and thereby
to reduce side effects.
[0043] Data obtained from cell culture assays and animal studies
can be used in formulating a range of dosages for use in humans.
The dosage of such compounds lies preferably within a range of
circulation concentrations that include the ED.sub.50 with little
or no toxicity. The dosage may vary within this range depending
upon the dosage form employed and the route of administration
utilized. For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC.sub.50 (i.e., the concentration of the test compound which
achieves a half-maximal inhibition of symptoms) as determined in
cell culture. Such information can be used to more accurately
determine useful doses in humans. Levels in plasma may be measured,
for example, by high performance liquid chromatography.
[0044] Generally, the therapeutically effective amount of the
pharmaceutical compositions used herein will vary with the age of
the subject and condition, as well as the nature and extent of the
disease, all of which can be determined by one of ordinary skill in
the art. The dosage may be adjusted by the physician, particularly
in the event of any complication. A therapeutically effective
amount will typically vary from 0.01 mg/kg to about 1000 mg/kg,
preferably from about 0.1 mg/kg to about 200 mg/kg, and most
preferably from about 0.2 mg/kg to about 20 mg/kg.
[0045] The present invention encompasses active agents which
modulate or inhibit CXCR4 activity. An agent may, for example, be a
small molecule. For example, such small molecules include, but are
not limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds. It is understood that appropriate doses of small
molecules depend upon a number of factors within the knowledge of
the ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have.
[0046] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram). It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. Such appropriate doses may be determined using the
assays described herein. When one or more of these small molecules
is to be administered to an animal (e.g., a human) in order to
modulate expression or activity of a polypeptide or nucleic acid of
the invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0047] In certain embodiments of the invention, a modulator or
inhibitor of CXCR4 activity is administered in combination with
other agents (e.g., a small molecule), or in conjunction with
another, complementary treatment regime. Accordingly, the subject
may be treated, for example with a CXCR4 inhibitor, and further
treated with another anti-cancer agent.
[0048] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0049] The pharmaceutical composition of the invention can include
any pharmaceutically acceptable carrier known in the art. Further,
the composition can include any adjuvant known in the art, e.g.,
Freund's complete or incomplete adjuvant. Preparations for
parenteral administration include sterile aqueous or non-aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils
such as olive oil, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, alcohol/aqueous solutions,
emulsions or suspensions, including saline and buffered media.
Parenteral vehicles include sodium chloride solution, Ringer's
dextrose, xylitol, dextrose and sodium chloride, lactated Ringer's
solution or fixed oils. Intravenous vehicles include fluid and
nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose or xylitol), and the like. Preservatives
and other additives may also be present such as, for example,
antimicrobials, antioxidants, chelating agents, inert gases and the
like.
[0050] The pharmaceutical compositions can be administered to the
mammal by any method which allows the CXCR4 inhibitor to reach the
appropriate bone marrow tissue or cells. These methods include,
e.g., injection, infusion, deposition, implantation, oral
ingestion, topical administration, or any combination thereof.
Injections can be, e.g., by intravenous, intramuscular,
intradermal, subcutaneous or intraperitoneal administration. Single
or multiple doses can be administered over a given time period,
depending upon the progression of the disease, as can be determined
by one skilled in the art without undue experimentation.
Administration can be alone or in combination with other
therapeutic agents. The route of administration will depend on the
composition of a particular therapeutic preparation of the
invention, and on the intended site of action. The present
compositions can be delivered directly to the site of action.
[0051] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the active compounds of the
invention, thereby increasing the convenience to the subject and
the physician. Many types of delayed release delivery systems are
available and known to those of ordinary skill in the art. These
include polymer-based systems such as polylactic and polyglycolic
acid, polyanhydrides and polycaprolactone; nonpolymer systems
include lipids such as sterols, and particularly cholesterol,
cholesterol esters and fatty acids or neutral fats such as mono-,
di- and triglycerides; hydrogel release systems; silastic systems;
peptide based systems; wax coatings, compressed tablets using
conventional binders and excipients, partially fused implants and
the like. In addition, pump-based hardware delivery systems can be
used, some of which are adapted for implantation.
[0052] A long-term sustained release implant also may be used.
"Long-term" release, as used herein, means that the implant is
constructed and arranged to deliver therapeutic levels of the
active ingredient for at least 30 days, and preferably 60 clays.
Long-term sustained release implants are well known to those of
ordinary skill in the art and include some of the release systems
described above.
[0053] With regard to both prophylactic and therapeutic methods of
treatment, such treatments may be specifically tailored or
modified, based on knowledge obtained from the field of
pharmacogenomics. "Pharmacogenomics", as used herein, refers to the
application of genomics technologies such as gene sequencing,
statistical genetics, and gene expression analysis to drugs in
clinical development and on the market. More specifically, the term
refers the study of how a patient's genes determine his or her
response to a drug (e.g., a patient's "drug response phenotype", or
"drug response genotype"). Pharmacogenomics thereby allows a
clinician or physician to target prophylactic or therapeutic
treatments to patients who will most benefit from the treatment and
to avoid treatment of patients who will experience toxic
drug-related side effects.
[0054] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, are incorporated herein by
reference.
[0055] In the following examples, a mouse model of multiple myeloma
is used. The murine model of is the 5T33MM model, which originated
from a spontaneous case of multiple myeloma in aged mice of the
C57BL/KaiwRij strain (Radl et al, 1979). The 5T33MM model is
initiated and maintained by transplanting multiple myeloma-affected
bone marrow into young syngenic mouse recipients. The clinical
characteristics of this mouse model are similar to human multiple
myeloma, which makes it a valuable tool to study multiple
myeloma.
EXAMPLE 1
Maintenance of 5T33MM Cells
[0056] 5T33MM cells can be maintained both in vivo and in vitro.
For in vivo maintenance, the bone marrow of sick mice is isolated
by flushing long bones (femora and tibia) with sterile phosphate
buffered saline (PBS). Flushing is performed with a syringe through
a 21G needle. After isolation, red blood cells are lysed and a
single cell suspension is prepared. These cells are injected in a
secondary recipient, which develops the disease within a certain
time period. The time for multiple myeloma development depends on
the number of cells injected. For in vitro maintenance, 5T33MM
cells are cultured in a special media, which includes RPMI-1640,
10% fetal calf serum (FCS), L-glutarnine, penicillin and
streptomycin, sodium pyruvate, non-essential amino acids, and
.beta.-mercaptoethanol. Cells are seeded in a culture flask, and
media is changed once in three days. Injection of the in vitro
cultured cells mediates disease development in recipient mice
within 2-4 weeks, depending on the number of cells injected. In
results shown, we only used in vitro cultured RT33MM cells.
EXAMPLE 2
CXCR4 Expression in Mouse 5T33MM Cells
[0057] Cells that home to the bone marrow typically utilize signals
from the chemokine receptor CXCR4 to undergo integrin mediated
arrest. 5T33MM cells express CXCR4, and it is hypothesized that the
development of multiple myeloma can be disrupted by interfering
with cell signaling through the CXCR4 receptor. Accordingly, a
virus that expresses the SDF1.alpha. degrakine is transduced into
5T33MM cells to reduce the expression of CXCR4 using retroviral
technology.
[0058] The production of the retrovirus is performed as described
in Li et al., J. Exp. Med., 189, pages 1399-1512 (1999), the
discolsure of which is incorporated by reference herein in its
entirety. 10 .mu.g of retroviral vector DNA and 5 .mu.g of
MCV-ecopac, an ecotropic single-genome packaging construct, are
transfected as per a 6-cm dish of HEK-293T as described in Finer et
al., Blood, 83, pages 43-50 (1994). The medium is changed at 24
hours, and a virus supernatant is harvested at 48 hours after
transfection. The supernatant is passed through a 0.45-.mu.m
filter, aliquoted, and frozen at 80.degree. C. 5T33 cells are
transduced with a retrovirus to improve the transduction efficiency
(Kotani et al., Human Gene Therapy, 5, pages 19-28 (1994)).
1.times.10.sup.6 5T33 cells and viral supernatant are
co-centrifuged at 700.times. g for 24 hours, and then moved to a
37.degree. C. incubator. Transduction efficiency is assayed by FACS
measurement of fluorescent reporter genes (eGFP and DsRed).
Transduced 5T33 cells are FACS sorted (FACS Aria, B&D) to
obtain homogeneous eGFP.sup.+ or DsRed.sup.+ populations of cells.
The dis
[0059] As shown in FIG. 1A, the vector-transduced cells express
CXCR4, while FIG. 1B shows that degrakine transduced cells express
60% less CXCR4.
EXAMPLE 3
Bone Marrow Homing of Mouse 5T33MM Cells
[0060] 5T33MM cells, obtained as described above, are injected into
recipient mice. While homing (2 hours) and retention (24 hours)
does not depend on CXCR4 expression, later events, such as cell
proliferation and/or survival (1 to 3 weeks) are decreased in the
absence of CXCR4 (FIG. 2).
[0061] Similar results are obtained when 5T33MM cells are
manipulated pharmaceutically instead of genetically: when
recepients are treated with the CXCR4 inhibitor AMD3100, it does
not affect the homing (2 hours) or retention (24 hours) of the
5T33MM cells, but it significantly reduces their
proliferation/survival (6 days) (FIG. 3).
EXAMPLE 4
Vector 5T33MM cells and SDF-1.alpha. Degrakine cells and Treatment
of Multiple Myeloma with AMD3100.
[0062] Following the injection of 10.times.10.sup.6 5T33.sup.vector
or 5T33.sup.SDF-DK cells, mice that received 5T33.sup.SDF-DK cells
displayed dramatically increased survival compared to mice injected
with 5T33.sup.vector cells. See FIG. 4.
[0063] When AMD3100 is administered at the time 5T33 cells are
injected, an increase in survival is observed. AMD3100 is delivered
via a subcutaneous osmotic pump at a concentration of 25 mg/mL and
a delivery rate of 0.5 .mu.l/hr. The osmotic devices delivered the
drug for 21 days. See FIG. 5.
[0064] Table 1 below shows the survival and serum IgG.sub.2b levels
of mice which were given 5T33 MM cells. The mice were transduced
with a control vector, the SDF1.alpha. degradine, or with twice the
levels of the SDF1.alpha. degradine vector. Mice that received
trasnduced 5T33MM cells experienced a reduction in mortality as
compare to the control mice. Mice that survived longer than 40 days
were considered free of the disease. In humans, multiple myeloma is
associated with increased levels of serum immunogobulin. Mice that
did not develop multiple myeloma do not have increased levels of
IgG.sub.2b paraprotein, while mice with multiple myeloma had
elevated IgG.sub.2b levels. TABLE-US-00001 TABLE 1 Survival (days
post 5T33 Serum IgG.sub.2b Levels injection) (mg/mL) Vector 1
>40 3.8 Vector 2 25 8.8 Vector 3 19 9.9 Vector 4 21 4.4 Vector 5
21 4.5 SDF1.alpha. Degrakine 1 >40 2.7 SDF1.alpha. Degrakine 2
>40 3.2 SDF1.alpha. Degrakine 3 >40 1.6
[0065] The above examples demonstrate, inter alia, that the
attenuation of CXCR4 expression in 5T33MM cells completely blocked
the development of fatal multiple myeloma in recipient mice even
when CXCR4 expression was only reduced to subnormal levels without
achieving complete inhibition of the receptor or preventing homing
to the bone marrow. Without wishing to be bound by any theory or
mechanism of action, it is believed that a strong signal sent
through the CXCR4 receptor is required for multiple myeloma
development, not only for hematogenous dissemination, but also for
the survivial and/or proliferation of bone marrow-resistant tumor
cells. Accordingly, it is concluded that interfering with the
function of the CXCR4 receptor on multiple myeloma cells can serve
as a method for treating multiple myeloma in humans.
[0066] A number of embodiments of the invention have been described
herein. Nevertheless, it will be understood that various
modifications may be made to the invention without departing from
its spirit and scope. Accordingly, embodiments other than those
specifically described herein are intended to be embraced by the
following claims. Those skilled in the art will be able to
ascertain, using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. These and all other equivalents are intended to be
encompassed by the following claims.
* * * * *